Early Endometriosis Liquid Biopsy Diagnosis with Immune System Derived Epigenetic Biomarker - Genome Profiling
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Early Endometriosis Liquid Biopsy
Diagnosis with Immune System
Derived Epigenetic Biomarker
New Research into the Untapped Potential
of Immune System Epigenetic Biomarkers
A White Paper by Adam Marsh, Ph.D.
Chief Science Officer and Co-Founder
Copyright © Genome Profiling, LLC, 2021 Page 0 of 11
Introduction In the U.S., 10 to 15 percent of women of reproductive age suffer from endometriosis. This disease is drastically under-diagnosed in early stages, resulting in decreased quality of life and severe complications due to advanced disease in millions of women. The current standard of care for diagnosis is laparoscopic biopsy of the lesions. However, the diffuse distribution of lesions in early stages, and the necessity of a skilled surgeon and pathologist to make accurate histological diagnoses, make this a difficult and expensive test to perform with high accuracy. In fact, many women with endometriosis undergo several laparoscopic procedures before a definitive diagnosis is reached. An accurate, blood-based diagnostic assay would establish an important advance in patient care, could be easily prescribed by patients’ primary care physicians, and would provide biopharma organizations with a new tool for use in clinical trials. Dan Martin, MD, Professor Emeritus, University of Tennessee Health Science Center, a life fellow of the American College of Obstetricians and Gynecologists, and current Scientific and Medical Director of the Endometriosis Foundation, observes, “Delayed diagnosis of early endometriosis is a major problem and opportunity. To date, endometriosis diagnostics have not been capable of reliably identifying early disease states even in specialized clinical settings without laparoscopic surgery. The undue suffering and pain that many patients routinely experience over years to arrive at a correct endometriosis diagnosis is staggering; not to mention the increased cost of multiple laparoscopic surgical procedures that could be saved. Recently, however, encouraging research advances link patient immune system status to endometriosis progression, and by leveraging immune cell epigenetic patterns in DNA methylation, there is an exciting potential for developing new clinical diagnostics. If this proves out, it will enable general clinicians to quickly screen for endometriosis and manage early disease medically or correctly refer their patients with nodules, cysts, or chronic symptoms to an appropriate endometriosis specialist for treatment. Ultimately, this would contribute to the development Copyright © Genome Profiling, LLC, 2021 Page 1 of 11
of a 'precision medicine' approach to endometriosis treatment and therapeutic drug development." Genome Profiling is proving that epigenetic DNA methylation shifts in immune system white blood cells (WBCs) are a prominent change caused by disease-specific stress that can be measured from simple whole blood samples. Identifying a set of such diagnostic "EpiMarkers" would revolutionize a general clinician’s ability to definitively identify endometriosis in patients, and would also reduce the overall health care costs currently expended on identifying this problematic disease. This white paper provides supportive literature and empirical evidence for the enormous potential of immune system derived epigenetic biomarkers, and how their translation into a precision blood test could transform the clinical standard of care for Endometriosis. Endometriosis and the Immune System Over the last three years, there has been growing mechanistic awareness of the interaction or response of the immune system to Endometriosis (see References for 2018-2021 publications). First, growing evidence points to specific immune suppression activity similar to that observed by aggressive tumors as they attempt to evade or suppress immune surveillance. Most notable is the increase in prevalence of T and B cells that are positive for PD-1 and PD-L1 expression in peripheral blood samples from patients with advanced endometriosis (see refs #12 & 16). This altered immune system state also involves shifts in IL-6, IL-8, TNF- alpha, and CLTA-4 expression in WBCs, which signals an overall inflammatory response both at the tissue disease site and systemically (see refs #1,4,6,8,9). In addition, functional changes in monocyte infiltration rates, macrophage behavior and natural killer cell activity indicate the large cellular differences in immune system state that accompany endometriosis disease progression (see refs #2,8,14,15). Copyright © Genome Profiling, LLC, 2021 Page 2 of 11
The interaction of the immune system with endometriosis
progression has been recently reviewed by Crispim et al.
(2021; see ref #5). They suggest that the range of potential immune
interaction mechanisms could account for the lack of a uniform
or well-established pathophysiology in all cases of endometriosis.
However, finding the most proximal immune/endometriosis linkage
could provide a druggable target that would be effective for
reducing the severity and progression of the disease in most cases.
Thus, much of the attention on identifying immune cellular and
molecular mechanisms/impacts of the disease is focused on finding
potential therapeutic targets (see refs #4,10,12,13,14). Although a
range of hormonal, genetic, immune and environmental factors are
known to play a role in the disease, the targeting of immune system
mechanisms is one of the most active areas of therapeutic drug
discovery.
Key Takeaways
• There is a strong impact and/or interaction between the
progression of endometriosis and altered immune system
activities, analogous to cancer tumor behaviors in terms of
evasion impairment and/or suppression of immune system
function.
• These immune system changes are not limited to the
infiltration of cells at the disease/lesion site, but rather are
evident in systemic immune system parameters associated
with peripheral blood circulation.
Precision Epigenetics is a Probe into Endometriosis Stress
Interestingly, there are a few recent endometriosis studies that focus
on genomic structure or architectural level changes (epigenetics)
that can produce large-scale shifts in cellular activities associated
with the disease (see refs #3,11). The authors of these studies focus
on chromosomal aberrations and differentially methylated DNA
domains (respectively) to identify large-scale organizational
changes that could result in altered gene expression. Thus,
Copyright © Genome Profiling, LLC, 2021 Page 3 of 11epigenetic processes are another important component of endometriosis, and this is the focus of applying Genome Profiling's Discovery and Analytics Platform to identify endometriosis epigenetic disease signals from standard peripheral whole blood draws. The sentinel specificity of circulating peripheral white blood cells (WBCs) is finely tuned to detect subtle abnormalities or stressors in tissue micro-environments (see Fig. 1). These cells respond to any perturbation from normal homeostasis by altering gene expression events to change cell surface signal receptors, intra-cellular signal transduction pathways, and signal ligand synthesis and export. These gene expression shifts are largely driven by epigenetic mechanisms, of which DNA methylation is one of the most prominent. Through this process, extracellular forces, such as differential immune cell changes arising from endometriosis disease stressors, become ”imprinted” on the genomes of circulating WBCs. Copyright © Genome Profiling, LLC, 2021 Page 4 of 11
GenPro's novel liquid biopsy assay and machine-learning (ML)
platform leverages this imprinting process to discover and quantify
differential, whole genome, WBC DNA methylation by CpG site.
From this data, the platform identifies the 20 to 60 CpG sites which
together form the strongest predictive CpG network, or signal, for
discriminating patient phenotypes that are specific to disease
presence, progression, or sub-groups. We call these networked sites
CpG-based epigenetic biomarkers, or “EpiMarkers.”
GenPro's capabilities are enabled by its proprietary integrated
science and machine-learning platform and methods, which make
novel EpiMarker discoveries and perform blind validations within
approximately 10 weeks. EpiMarker discoveries are followed by
EpiMarker translations into a high-throughput target panel assay
for routine use on standard lab equipment within approximately
16 weeks.
The classification performance of GenPro's ML method generates
results that are superior to commercial chip assays. These results
are achieved using a Methyl-Sensitive Restriction Enzyme Method
(MSRE) during the EpiMarker discovery phase, rather than the
traditional bisulfite oxidation approach, resulting in higher-resolution
of genome-wide DNA methylation data.
The proprietary ML-based algorithms used for classification and
prediction are optimized for analyzing DNA methylation data.
The “prediction engine” used for every GenPro EpiMarker-enabled
assay has been engineered over the past five years and is readily
adaptive to nearly any disease or drug opportunity.
Key Takeaways
• Epigenetics is a new and emerging frontier in endometriosis
disease research.
Copyright © Genome Profiling, LLC, 2021 Page 5 of 11• GenPro’s technology is an exciting novel application of
immune-methylome profiling of WBCs obtained from simple
whole blood liquid biopsies.
Proof-of-Concept Results:
GenPro has recently executed a proof-of-concept study with a
clinical reproductive health center. The analyzed subject cohort
consisted of 15 normal non-endometriosis patients, 15 patients that
had been diagnosed with ectopic endometriosis, and 15 patients
with early stage (eutopic) endometriotic lesions. An EpiMarker
discovery and blind validation study was performed using tissue
from these patients with subsequent follow-on comparisons to
blood samples obtained from a smaller subset of five patients from
each of the three groups. The resultant EpiMarker-enabled immune
cell methylome discrimination between the groups was highly
significant (see Fig. 2). There is a remarkably strong signal in the CpG
site methylation profiles across a broad number of genes.
Figure 2. NMDS, Ordinate Analysis Plane. The three
patient groups are separated by their DNA methylation
profiles. Each point is a single patient; ellipses show 90%
confidence interval.
Copyright © Genome Profiling, LLC, 2021 Page 6 of 11Using GenPro's proprietary machine-learning platform, the
phenotypic discrimination between the healthy controls and the
ectopic patients (control vs. invasive) was distilled down to a core
EpiMarker set consisting of 50 predictive CpG sites. This multi-CpG-
site EpiMarker was then applied to a blind validation strategy with
a k-fold cross validation analysis of predictive performance, which
yielded an overall accuracy of 93 percent, with just one false
positive and one false negative out of 30 total samples. This high
degree of predictive classification indicates the strong difference
in epigenetic methylome signals that GenPro's technology can
identify.
Although the accompanying blood samples were too few to
execute a classification analysis, the differential separation in
an ordinate analysis (as in Fig. 2) confirms that there is a large
difference in whole blood immuno-methylome profiles as well.
In addition, GenPro's Analytics yielded functional insights across
genes, pathways and genome structure/domains. Of particular
interest in endometriosis disease etiology is an apparent linkage
to estrogen response (see refs #4,7). In GenPro's study, it was
absolutely clear that the estrogen receptor 1 gene (ESR1) was
hypermethylated in the disease patient group (see Fig. 3). With
GenPro's site-specific CpG resolution, a strong shift in methylation
in the endometriosis patients was particularly evident near the
transcriptional start site, in the first to third exons, and prominently
at the 3' UTR terminus of the gene.
Figure 3. Differential Methylation
by CpG site across the Estrogen
Receptor 1 gene (ESR1). The dMET
score is calculated as the difference
in %methylation at each site
calculated as CON minus INV
(ectopic). Dotted line indicates
the Transcriptional start site and NT
position is relative to TSS = 0.
Copyright © Genome Profiling, LLC, 2021 Page 7 of 11Key Takeaways
• For a small POC study, the attainment of 93-percent
prediction accuracy straight out of the gate is a strong
indication of the likely success of pursuing a fully developed
clinical assay.
• GenPro's CpG methylation measurements have functional
implications for assessing the relevance of any observed shifts
as with the ESR1 gene in endometriosis patients.
• GenPro's immuno-methylome profiling of peripheral whole
blood can serve as a simple clinical diagnostic tool for
identifying early endometriosis, and potentially for describing
immunologic mechanisms contributing to disease symptom
severity/progression.
Summary
Different endometriosis stressors likely lead to different immune
system responses. The methylomes of WBCs are imprinted with the
unique disease stressors encountered in an endometriosis patient.
Thus, determining the epigenetic profile of WBCs is a highly efficient
approach to identify endometriosis disease, and perhaps to
establish a scale of the disease’s severity.
For general clinicians who are treating women with pelvic pain or
other symptoms related to reproduction and fertility, GenPro’s
capability to develop an EpiMarker and translate it into a targeted
panel screening assay can provide a rapid minimally invasive
diagnosis. A GenPro targeted liquid biopsy assay would utilize whole
blood samples, identify those patients in need of laparoscopy, and
allow those without endometriosis to avoid an invasive and costly
procedure.
Copyright © Genome Profiling, LLC, 2021 Page 8 of 11For the new wave of immune system targeted drugs in development, GenPro's technology could easily be applied to identify immune cell markers for patients most likely to respond to specific therapeutic drugs. Even retrospective analyses of completed clinical trials could provide highly valuable insights and enable a second “shot-on-goal,” by using the GenPro assay as an enrichment mechanism for a biomarker-grounded post-hoc efficacy analysis. Similarly, a GenPro EpiMarker assay could be implemented proactively in a trial, as a patient stratification mechanism to establish trial arms based on each patient’s likelihood of response. All components for an EpiMarker Project to screen or stratify endometriosis patients have been vetted by GenPro POC studies to date (Fig. 2 and 3). GenPro EpiMarkers are a compelling diagnostic biomarker solution with high relevance to clinical trial use for therapeutic drug development. When used for making definitive diagnoses, they offer significant potential for improved patient care and outcomes, as well as large reductions in medical costs associated unneeded procedures, repeat office visits, and treating complications related to advanced diseases. When used to stratify patients entering a clinical trial, these biomarkers could increase the likelihood of demonstrating drug efficacy, and lead to more successful trial outcomes. Copyright © Genome Profiling, LLC, 2021 Page 9 of 11
About the Author: Adam G. Marsh, Ph.D. adam.marsh@genomeprofiling.com Adam is an Associate Professor, at the University of Delaware, and a Co-Founder and the CSO at Genome Profiling, LLC. At the University of Delaware, he is a faculty member with appointments in the Center for Bioinformatics and Computational Biology as well as the School of Marine Science. His academic research career has been supported primarily by grants from the National Science Foundation. From 2016 to 2020 he served as Senior Research Associate at the Helen F. Graham Cancer Center, Newark, DE, an adjunct appointment in support of Genome Profiling's research on DNA methylation markers of early breast cancer risk (DCIS; ductal carcinoma in situ). Adam has published over 65 papers, including first authorship on Science and Nature papers. https://scholar.google.com/citations?hl=en&user=Qfm6-5gAAAAJ References (relevant selections from 2018 to 2021) 1. Abramiuk, M., Bebnowska, D., induced chromosomal aberrations in Hrynkiewicz, R., Niedzwiedzka-Rystwej, P., peripheral blood of women with Polak, G., Kotarski, J., et al. (2021). Clta-4 endometriosis: evidence of genomic expression is associated with the instability. MINERVA OBSTETRICS AND maintenance of chronic inflammation in GYNECOLOGY 73, 369–375. endometriosis and infertility. CELLS 10, 112– doi:10.23736/S2724-606X.21.04773-4 118. doi:10.3390/cells10030487 4. Burns, K., Thomas, S., Hamilton, K., Young, 2. Bedient, C., Rodriguez, D., Sidell, N., S., Cook, D., and Korach, K. (2018). Early en- Roberts, C., and Schutte, S. (2019). dometriosis in females is directed by Alterations in the inva- sive properties of immune-mediated estrogen receptor alpha peripheral blood monocytes from patients and il-6 cross-talk. ENDOCRINOLOGY 159, with endometriosis. CLINICAL AND 103–118. doi:10.1210/en.2017-00562 EXPERIMENTAL OBSTETRICS & GYNECOLOGY 5. Crispim, P., Jammal, M., Murta, E., and 46, 60–65. doi:10.12891/ceog4333.2019 Nomelini, R. (2021). Endometriosis: What is 3. Borjian Boroujeni, P., Beheshti, Z., Eshaghi the influence of immune cells? Zadeh, S., Afsharian, P., and Mohseni IMMUNOLOGICAL INVESTIGATIONS 50, 372– Meybodi, A. (2021). Spontaneous and 388. doi: 10.1080/08820139.2020.1764577 Copyright © Genome Profiling, LLC, 2021 Page 10 of 11
6. Guo, M., Bafligil, C., Tapmeier, T., 12. Oksasoglu, B., Hepokur, C., Misir, S., Yildiz, Hubbard, C., Manek, S., Shang, C., et al. C., Sonmez, G., and Yanik, A. (2021). (2020). Mass cytometry analysis reveals a Determination of pd-1 expression in distinct immune environment in peritoneal peripheral blood cells in patients with fluid in endometriosis: a characterisation endometriosis. GYNECOLOGICAL study. BMC MEDICINE 18, 272–283. ENDOCRINOLOGY 37, 157–161. doi:10.1186/s12916-019-1470-y doi:10.1080/09513590.2020.1821358 7. Jedryka, M., Chrobak, A., Chelmonska- 13. Patel, B., Lenk, E., Lebovic, D., Shu, Y., Yu, Soyta, A., Fijalkowska, D., and Matkowski, R. J., and Taylor, R. (2018). Pathogenesis of (2020). Decreased expression of estrogen endometriosis: Interaction between receptors alpha and beta in peripheral endocrine and inflammatory pathways. BEST blood lymphocytes from the endometrial PRAC- TICE & RESEARCH CLINICAL cancer patients and women with OBSTETRICS & GYNAECOLOGY 50, 50–60. endometriosis. EUROPEAN JOURNAL OF doi: 10.1016/j.bpobgyn.2018.01.006 GYNAECOLOGICAL ONCOLOGY 41, 375– 14. Vallve-Juanico, J., Houshdaran, S., and 379. doi:10.31083/j.ejgo.2020.03.5054 Giudice, L. (2019). The endometrial immune 8. Jerman, L., Anderson, L., Markham, R., environ- ment of women with endometriosis. and Hey-Cunningham, A. (2020). The HUMAN REPRODUCTION UPDATE 25, 565– lymphatic system in endometriosis: a pilot 592. doi: 10.1093/humupd/dmz018 study of endometrial-like cells and immune 15. Villanacci, R., Bandini, V., Ottolina, J., cell populations in lymph nodes associated Pagliardini, L., Candiani, M., and Vigano, P. with deep infiltrating bowel lesions. (2021). The pathogenesis of endometriosis: REPRODUCTIVE SCIENCES 27, 977– 987. clues from the immunological evidence. doi:10.1007/s43032-020-00171-0 MINERVA OBSTETRICS AND GYNECOLOGY 9. Leenen, S., Hermens, M., van Steenwijk, P., 73, 275–282. doi:10.23736/S2724- Bekkers, R., and van Esch, E. (2021). 606X.21.04768-7 Immunologic factors involved in the 16. Walankiewicz, M., Grywalska, E., Polak, malignant transformation of endometriosis G., Korona-Glowniak, I., Witt, E., Surdacka, to endometriosis-associated ovarian A., et al. (2018). The increase of circulating carcinoma. CANCER IMMUNOLOGY pd-1-and pd-l1-expressing lymphocytes in IMMUNOTHERAPY 70, 1821–1829. doi: endometriosis: Correlation with clinical and 10.1007/s00262-020-02831-1 laboratory parameters. MEDIATORS OF 10. Miller, J., Symons, L., Marks, R., and INFLAMMATION 2018. Tayade, C. (2020). Endometrial Immune- doi:10.1155/2018/7041342 Inflammatory Gene Signatures in Endometriosis (Springer, Cham.). doi:10.1007/978-3-030-28584-5 10 11. Mortlock, S., Restuadi, R., Levien, R., Girling, J., Holdsworth-Carson, S., Healey, M., et al. (2019). Genetic regulation of methylation in human endometrium and blood and gene targets for reproductive diseases. CLINICAL EPIGENETICS 11. doi:10.1186/s13148-019-0648-7 Copyright © Genome Profiling, LLC, 2021 Page 11 of 11
We'd love to talk. Contact us today to discuss how GenPro can provide a novel, immune-system-derived, epigenetic biomarker and liquid biopsy assay tailored to your drug for precision identification of responder and non-responder patients. Contact us today: adam.marsh@genomeprofiling.com (302)-249-5458 www.genomeprofiling.com Copyright © Genome Profiling, LLC, 2021 Page 11 of 11
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